[unreadable] Dr. Ray A. Caldwell is proposing original research for understanding the physiological regulation of the epithelial sodium channel (ENaC) by serine proteases (SPs). The recent discovery of endogenous epithelial SPs and their effects on ENaC reveal a novel and potentially important pathway for epithelial Na+transport. The candidate probes the effects of SPs on ENaC at the most basic level, the single channel, and provides direct evidence for an elusive subset (i.e. silent) of ENaCs that exhibit a robust increase in channel open probability (Po) when acted on by low concentrations of SP. To elucidate the mechanism(s) by which SPs increase ENaC-mediated currents, Dr. Caldwell will test the following hypothesis: SPs increase EnaC Po through proteolytic cleavage of an extracellular gating regulatory domain. To achieve this objective, he will use the patch-clamp technique to address 3 specific aims: 1) to determine the mechanism of action by characterizing the effect(s) of SPs on ENaC, 2) to elucidate the putative ENaC gating domain by engineering mutations in the extracellular loop of the protein that renders the channel insensitive to SPs, and 3) to test the role of these protease-sensitive gating domains in epithelial models of ENaC function. Experimental results from these studies are anticipated to have an immediate impact on existing models of ENaC regulation. For instance, Dr. Caldwell's recently published work challenges the view that ENaC, residing at the cell surface membrane, is constitutively active, and regulated only through endo/exocytosis. Results from the experiments described will be used to provide a more comprehensive model of epithelial Na+transport as well as potentially novel therapeutic approaches to diseases involving ENaC, such as cystic fibrosis and hypertension. The KO1 award will provide the foundation for Dr. Caldwell to pursue his career goals of establishing scientific funding and continued publication of research results in peer-reviewed journals. Collectively, these components will be vital for him to emerge as an independent investigator [unreadable] [unreadable]